Effects of a Disulfide Crosslink (XL) on the Trypsin Cleavage Pattern of Rabbit Cardiac Tropomyosin (TM)

Abstract

A specific tryptic cleavage site at R133 with a subsequent site at R182 in Tm (1-284) was identified by Pato, et al, 1981. Ueno reported (1984) that a disulfide XL at C190 increased the rate of cleavage at higher temperatures (35o-40o). Here we redetermined the affect of the XL on the cleavage pattern at 26o, where Tm is >95% helical, and at 40o, where the middle region of Tm is unfolded. We used SDS-gels, N-terminal sequencing, and MALDI, to identify the products of cleavage. We found that the rate of Tm cleavage at R133 was unaffected by the crosslink at 26oC, but was slightly increased at 40oC. XL cleavage intermediates containing one cleaved and one uncleaved chain were observed. At 26o, cleavage of XL Tm at R133 produced a 51K XL heterodimer, (134-284/1-284), and a 35K XL homodimer, (134-284)2. At longer times, a second cleavage site at R168, produced a smaller 27K XL homodimer (169-284)2. At 40o, The XL increased the cleavage rate at K168 to produce a 47K XL heterodimer (169-284/1-284) and the 51K XL heterodimer. A shorter 31K XL heterodimer, (169-284/134-284) was also produced. The homodimer, (169-284)2 was also present early . The 35K homodimer seen at 26o was not produced. This study detected cleavage intermediates that could not be seen for uncrosslinked Tm. A new cleavage at K168 was seen which dominated at 40o. Cleavage at R183 after the initial cleavage at R133, (Pato et al, 1981), was not observed. This shows that: 1) both chains are not cleaved simultaneously; 2) locally unstable regions near R133 and R168 exhibit increased flexibility especially at physiological temperatures. Supported by NIH HL 22461-29 and an ARRA student summer grant. to D.Y.